EP4071242A1 - Amylasepolypeptide mit verbesserten eigenschaften - Google Patents

Amylasepolypeptide mit verbesserten eigenschaften Download PDF

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Publication number
EP4071242A1
EP4071242A1 EP21167053.4A EP21167053A EP4071242A1 EP 4071242 A1 EP4071242 A1 EP 4071242A1 EP 21167053 A EP21167053 A EP 21167053A EP 4071242 A1 EP4071242 A1 EP 4071242A1
Authority
EP
European Patent Office
Prior art keywords
seq
amylase
variant
amino acid
active fragment
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP21167053.4A
Other languages
English (en)
French (fr)
Inventor
Steven Doig
Ryan FRISCH
Svend HAANING
Frank Koopman
Karsten Matthias Kragh
Lene KRAGH
Chris Leeflang
Sina Pricelius
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
DuPont Nutrition Biosciences ApS
Original Assignee
DuPont Nutrition Biosciences ApS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by DuPont Nutrition Biosciences ApS filed Critical DuPont Nutrition Biosciences ApS
Priority to EP21167053.4A priority Critical patent/EP4071242A1/de
Priority to CN202280032528.XA priority patent/CN117769596A/zh
Priority to EP22718025.4A priority patent/EP4320230A1/de
Priority to PCT/US2022/023637 priority patent/WO2022216801A1/en
Publication of EP4071242A1 publication Critical patent/EP4071242A1/de
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
    • A—HUMAN NECESSITIES
    • A21—BAKING; EDIBLE DOUGHS
    • A21D—TREATMENT, e.g. PRESERVATION, OF FLOUR OR DOUGH, e.g. BY ADDITION OF MATERIALS; BAKING; BAKERY PRODUCTS; PRESERVATION THEREOF
    • A21D8/00—Methods for preparing or baking dough
    • A21D8/02—Methods for preparing dough; Treating dough prior to baking
    • A21D8/04—Methods for preparing dough; Treating dough prior to baking treating dough with microorganisms or enzymes
    • A21D8/042—Methods for preparing dough; Treating dough prior to baking treating dough with microorganisms or enzymes with enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
    • C12N9/2414—Alpha-amylase (3.2.1.1.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
    • C12N9/2414—Alpha-amylase (3.2.1.1.)
    • C12N9/2417—Alpha-amylase (3.2.1.1.) from microbiological source
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/0106—Glucan 1,4-alpha-maltotetraohydrolase (3.2.1.60)
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/06—Enzymes

Definitions

  • amylopectin In the dough most of the amylopectin is found inside the starch granules in a crystalline form. During baking the starch granules will take up water in a process called gelatinization and some of the amylopectin will leak out of the starch granule. In the freshly baked bread, most of the amylopectin will no longer be in the crystalline form but rather form an amorphous network throughout the bread. Over time the amylopectin will recrystallize, a process called retrogradation, thereby forming a stronger network which in turn lead to the decrease in bread softness and resilience associated with bread staling. The speed and extent of amylopectin retrogradation is dependent on the length of amylopectin sidechains with shorter sidechains slowing the retrogradation.
  • a process for treating starch by contacting starch with a PS4 variant or an amylase active fragment thereof as described above and allowing the PS4 variant or an amylase active fragment thereof to generate from the starch one or more linear products.
  • a process for making a bakery product having the steps of (a) providing a starch medium; (b) adding to the starch medium a PS4 variant or an amylase active fragment thereof as described above; and (c) applying heat to the starch medium during or after step (b) to produce a bakery product.
  • the exoamylase is a maltogenic amylase.
  • the maltogenic amylase is a polypeptide according to SEQ ID NO:24.
  • the exoamylase is a non-maltogenic amylase.
  • the additional enzyme is a phospholipase or a galactolipase.
  • protein includes proteins, polypeptides, and peptides.
  • amino acid sequence is synonymous with the term “polypeptide” and/or the term “protein”.
  • the name of the amino acid the conventional one-letter and three-letter codes for amino acid residues may be used.
  • the 3-letter code for amino acids as defined in conformity with the IUPACIUB Joint Commission on Biochemical Nomenclature (JCBN). It is also understood that a polypeptide may be coded for by more than one nucleotide sequence due to the degeneracy of the genetic code. Unless otherwise indicated, any amino acid sequences are written left to right in amino to carboxy orientation.
  • the present invention encompasses variants, homologues, derivatives and fragments thereof.
  • isolated variant or isolated amylase active fragment thereof as used herein refers to a polypeptide which is at least 30% pure, at least 40% pure, at least 60% pure, at least 80% pure, at least 90% pure, and at least 95% pure, as determined by SDS-PAGE.
  • a homologue of a PS4 variant according to the present invention is an active amylase.
  • the homologue of a PS4 variant according to the present invention has amylase activity.
  • the present invention provides a method for improving at least one PI value as described herein, such as a baking PI value in a dough, dough product, processed dough product, or a bakery product.
  • a constitutive promoter was fused to the 5' end of the signal peptide sequence, and an antibiotic resistance marker DNA cassette together with 3' and 5'-end flanking DNA regions homologous to Bacillus licheniformis were added to the expression cassettes as well, enabling homologous recombination and selective growth of Bacillus licheniformis transformants.
  • These DNA cassettes were transformed into Bacillus licheniformis cells using methods known in the art. Transformants were grown on selective agar plates and in selective liquid medium by making use of the appropriate antibiotic resistance selection. For each individual clone, DNA sequence encoding the amylase variant expression cassette was PCR amplified and analysed by Sanger sequencing to identify the DNA sequence of the amylase variant.
  • E-MALTS E-MALTS in 50 mM Na-Acetate, 5 mM CaCl 2 , 0.001% Triton, pH 5.5 was mixed with 65 ⁇ L 50 mM Na-Acetate, 5 mM CaCl 2 , 0.001% Triton, pH 5.5 and 10 ⁇ L enzyme solution in a MTP. Then the plate was incubated at 30C for 15 min. Next 150 ⁇ L 4% TRIS base was added and OD 410 nm was measured.
EP21167053.4A 2021-04-06 2021-04-06 Amylasepolypeptide mit verbesserten eigenschaften Withdrawn EP4071242A1 (de)

Priority Applications (4)

Application Number Priority Date Filing Date Title
EP21167053.4A EP4071242A1 (de) 2021-04-06 2021-04-06 Amylasepolypeptide mit verbesserten eigenschaften
CN202280032528.XA CN117769596A (zh) 2021-04-06 2022-04-06 具有改善的性质的淀粉酶多肽
EP22718025.4A EP4320230A1 (de) 2021-04-06 2022-04-06 Amylasepolypeptide mit verbesserten eigenschaften
PCT/US2022/023637 WO2022216801A1 (en) 2021-04-06 2022-04-06 Amylase polypeptides with improved properties

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
EP21167053.4A EP4071242A1 (de) 2021-04-06 2021-04-06 Amylasepolypeptide mit verbesserten eigenschaften

Publications (1)

Publication Number Publication Date
EP4071242A1 true EP4071242A1 (de) 2022-10-12

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EP21167053.4A Withdrawn EP4071242A1 (de) 2021-04-06 2021-04-06 Amylasepolypeptide mit verbesserten eigenschaften
EP22718025.4A Pending EP4320230A1 (de) 2021-04-06 2022-04-06 Amylasepolypeptide mit verbesserten eigenschaften

Family Applications After (1)

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EP22718025.4A Pending EP4320230A1 (de) 2021-04-06 2022-04-06 Amylasepolypeptide mit verbesserten eigenschaften

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EP (2) EP4071242A1 (de)
CN (1) CN117769596A (de)
WO (1) WO2022216801A1 (de)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024088549A1 (en) 2022-10-24 2024-05-02 Novozymes A/S Baking method with thermostable amg variant and alpha-amylase
WO2024088550A1 (en) 2022-10-24 2024-05-02 Novozymes A/S Baking method for pulse protein fortified bread employing thermostable amyloglucosidase variante (ec 3.2.1.3)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006003461A2 (en) * 2004-07-07 2006-01-12 Danisco A/S Polypeptide
WO2007007053A1 (en) * 2005-07-07 2007-01-18 Danisco A/S Modified amylase from pseudomonas saccharophilia
WO2010133644A2 (en) * 2009-05-19 2010-11-25 Danisco A/S Amylase polypeptides
WO2020185737A1 (en) * 2019-03-11 2020-09-17 Basf Se Amylases and methods for making and using them

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006003461A2 (en) * 2004-07-07 2006-01-12 Danisco A/S Polypeptide
WO2007007053A1 (en) * 2005-07-07 2007-01-18 Danisco A/S Modified amylase from pseudomonas saccharophilia
WO2010133644A2 (en) * 2009-05-19 2010-11-25 Danisco A/S Amylase polypeptides
WO2020185737A1 (en) * 2019-03-11 2020-09-17 Basf Se Amylases and methods for making and using them

Non-Patent Citations (12)

* Cited by examiner, † Cited by third party
Title
"Oligonucleotide Synthesis: A Practical Approach", 1984, IRI PRESS
ALTSCHUL ET AL., J. MOL. BIOL., 1990, pages 403 - 410
AUSUBEL ET AL., SHORT PROTOCOLS IN MOLECULAR BIOLOGY, 1999
AUSUBEL, F. M. ET AL.: "Current Protocols in Molecular Biology", 1995, JOHN WILEY & SONS
B. ROEJ. CRABTREEA. KAHN: "DNA Isolation and Sequencing: Essential Techniques", 1996, JOHN WILEY & SONS
D. M. J. LILLEYJ. E. DAHLBERG: "Methods of Enzymology: DNA Structure Part A: Synthesis and Physical Analysis of DNA Methods in Enzymology", 1992, ACADEMIC PRESS
FEMS MICROBIOL LETT, vol. 177, no. 1, 1999, pages 187 - 8
FEMSMICROBIOL LETT, vol. 174, no. 2, 1999, pages 247 - 50
HIGGINS DG & SHARP PM, GENE, vol. 73, no. 1, 1988, pages 237 - 244
HORWELL DC, TRENDS BIOTECHNOL, vol. 13, no. 4, 1995, pages 132 - 134
J. SAMBROOKE. F. FRITSCHT. MANIATIS: "Molecular Cloning: A Laboratory Manual", 1989, COLD SPRING HARBOR LABORATORY PRESS
SIMON RJ ET AL., PNAS, vol. 89, no. 20, 1992, pages 9367 - 9371

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Publication number Publication date
CN117769596A (zh) 2024-03-26
EP4320230A1 (de) 2024-02-14
WO2022216801A1 (en) 2022-10-13

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